DESCRIPTION: The long-term objective of this work is to understand the mechanisms underlying calcium-mediated exocytosis of neurotransmitter during synaptic transmission. Chemical synapses are the targets of many toxins, therapeutic drugs, and drugs of abuse that affect the brain by altering synaptic transmission. Plasticity of chemical synaptic transmission underlies some forms of learning and memory, and disorders of specific neurotransmitter systems underlie a number of psychiatric, neurological, and neuromuscular diseases. A better understanding of synaptic transmission will thus advance human health. The proposed experiments will investigate the presynaptic physiology of hair cells from the frog saccules. Hair cells are the specialized sensory receptors of the auditory and vestibular systems. Their unique combination of anatomical and physiological properties make them ideally suited for studies of presynaptic mechanisms. These properties include: (a) individual hair cells can be isolated and studied in vitro; (b) their compact shape allows rapid control of the membrane potential at synapses, using the whole-cell and perforated-patch methods; (c) the presynaptic calcium current can easily be separated from other currents and recorded in voltage clamp experiments; (d) the presynaptic calcium concentration can be estimated from the activity of presynaptic Kca channels; (e) capacitance measurements can be used to monitor exocytosis; (f) known concentrations of exogenous substances can be rapidly added to the cytoplasm through the recording pipette. These features will allow a detailed analysis of the roles of calcium-binding proteins (calbindin-D28k and synaptotagmin) in synaptic transmission. The specific aims of the project are: (1) To test the hypothesis that calbindin-D28k influences short-range calcium signaling ion frog saccular hair cells. (2) To optimize the method for measuring rapid changes in membrane capacitance. (3) To test the hypothesis that depolarization-evoked increases in membrane capacitance are due to the fusion of synaptic vesicles with the plasma membrane at active zones. (4) To test the hypothesis that only the synaptic vesicles that lie alongside the rows of presynaptic calcium channels are available for immediate release, and that the maximum sustainable exocytotic rate is limited by the rate at which this release-ready pool can be supplied from other pool(s) of vesicles. Morphological studies using fluorescence microscopy and electron microscopy will be used to correlate the physiological measurements with synaptic structures. These studies will provide new, quantitative information about calcium signaling and neurotransmitter release that should be applicable to other synapses in the nervous system.